'Where it all begins'... the great awakening', these are some of the terms that have been used to describe egg activation at fertilization. They highlight the fascination surrounding the subject. Indeed, the activation of development at fertilization is one of the most intriguing areas of reproductive and developmental biology, because of its medical, social and economic implications. Ca2+ is the universal signal for egg activation at fertilization in all sexually reproducing species, from plants to humans. Importantly, the fertilization-induced Ca2+ signal has specialized spatial and temporal kinetics that are vital for egg activation and the initiation of embryogenesis. Eggs acquire the ability to produce this specialized Ca2+ signal during oocyte maturation. However, despite the fundamental role of Ca2+ signaling differentiation its molecular regulation remains poorly understood. This proposal aims to elucidate the molecular mechanisms regulating the differentiation of Ca2+ signaling pathways during oocyte maturation in Xenopus. Xenopus offers an exceptional system for the proposed studies, because it is a good model for vertebrate fertilization, and because the Xenopus oocyte is a favored cell to study Ca2+ signaling. Fertilization in Xenopus induces a sustained Ca2+ rise that lasts for several minutes, and is essential for egg activation. Our Preliminary Studies show that the two most important determinants of the sustained Ca2+ rise in eggs are: prolonged opening of IP3 receptors, & removal of the plasma membrane Ca2+-ATPase (PMCA) from the cell membrane. Therefore, defining the regulation of these two Ca2+ effectors during oocyte maturation becomes essential to our understanding of how Ca2+ signals differentiate during maturation in preparation for fertilization. Studies proposed in Aim 1 will determine whether the prolonged opening of IP3 receptors is due to phosphorylation by MPF, using functional Ca2+ imaging, phosphopeptide mapping and patch clamping approaches. Aim 2 proposes experiments that will elucidate the mechanisms mediating PMCA endocytosis during oocyte maturation. Approaches to be used include functional knockdown, mass spectrometry and structure-function mutational analyses. Together the proposed studies will greatly improve our understanding of how eggs modulate Ca2+ signaling effectors to produce the required specialized Ca2+ transient at fertilization. Such knowledge is fundamentally important because the fertilization-induced Ca2+ transient is essential for egg activation, which defines the developmental competence of the embryo. [unreadable] [unreadable] [unreadable]